Glaucoma is an optic neuropathy characterized by retinal ganglion cell (RGC) death and optic nerve degeneration. Unfortunately, there are no current treatments that specifically target neurodegeneration. Identifying the signaling pathways responsible for disease onset and progression will be an important step in developing effective pharmacologic interventions for glaucomatous neurodegeneration. The endothelin (EDN) system is widely expressed throughout the body and participates in both physiological and pathophysiological processes. There is now a large body of evidence implicating the EDN system in human and in animal models of glaucoma. Manipulation of the EDN system significantly lessens RGC loss in a genetic and two inducible models of glaucoma. Also EDN signaling is upregulated in both the retina and optic nerve head prior to any signs of RGC death or dysfunction in an ocular hypertensive model of glaucoma. Despite the potential importance of EDN in glaucomatous neurodegeneration, the molecular mechanisms of EDN-induced RGC death are completely undefined. In particular, the upstream regulators and the downstream effectors of the EDN system are not known. Contributing to the difficulty in understanding how EDN signaling plays a role in glaucomatous neurodegeneration is the fact that EDN receptors, Ednra and Ednrb, are expressed on numerous cell types in the retina and optic nerve head including RGCs, astrocytes, myeloid derived cells (microglia and macrophages), and mural cells (pericytes and smooth muscle vascular cells). Furthermore, EDN is known to affect all of these cells in ways that are consistent with pathological responses observed in glaucoma. Thus, in order to understand EDN signaling in glaucoma the requirement of components of the EDN system, both receptors and ligands, must be systematically tested in the glaucoma-relevant cells they are expressed in. Here, we will test the hypothesis that defining the role of the EDN system in RGC death will identify early, critical signaling pathways that underlie glaucoma pathogenesis. To accomplish this and to define how EDN signaling functions after glaucoma-relevant insults, we will 1) define the mechanisms within RGCs that are required for EDN-induced RGC death, 2) determine the receptor, cell type, and molecular pathway(s) controlling EDN-induced RGC death, and 3) determine which cell types and molecular pathways are responsible for producing pathogenic EDN ligands. Importantly, this application will focus on the role of the EDN system in individual cell types, conditionally removing EDN components and performing transcriptomics on individual cell types. Together, the experiments outlined in the application critically test a long-standing hypothesis about the role of EDN in glaucoma and identify the various cell types and molecular mechanisms controlling glaucoma-relevant pathogenic EDN signaling. Given the early nature and proven role of EDN signaling in glaucomatous neurodegeneration these experiments will identify novel targets for developing neuroprotective treatments for glaucoma at early stages of disease pathogenesis.